Abstract

2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17β-estradiol (E2) that arises from the hydroxylation and subsequent methylation at the 2-position. In vitro 2ME2 inhibits a large variety of tumor and nontumor cell lines from diverse origins, as well as several stages of the angiogenic cascade. In vivo it has been shown to be a very effective inhibitor of tumor growth and angiogenesis in numerous models. Although various molecular targets have been proposed for this compound, the mechanism of action is still uncertain. As this molecule emerges as a drug candidate it is important to assess the estrogen receptors (ERs) as molecular targets for 2ME2. The purpose of this study was to investigate whether 2ME2 is able to engage ERs as an agonist and whether its antiproliferative activities are mediated through ERs. We confirm that 2ME2 has a lower binding affinity for ERα as compared with E2 and other E2 metabolites and antagonists, and we demonstrate that the affinity of 2ME2 for ERβ is even lower. When assessed in the presence of galangin, a cytochrome P450 enzyme inhibitor, at concentrations at which 2ME2 interacts with ERα in an in vitro binding assay, it does not stimulate the proliferation of an estrogen-dependent breast carcinoma cell line. Similar IC50 values for inhibition of proliferation and induction of apoptosis are obtained in estrogen-dependent and estrogen-independent human breast cancer cell lines, irrespective of the expression of ERα and ERβ. Moreover, the estrogen antagonist ICI 182,780 does not inhibit the antiproliferative activity of 2ME2. In E2-responsive cells such as MCF-7 and human umbilical vascular endothelial cells, high levels of E2 inhibit the antiproliferative activity of ICI 182,780 but not of 2ME2. Collectively, these results suggest that 2ME2 is distinct among estradiol metabolites because of its inability to engage ERs as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of ERα and ERβ.

INTRODUCTION

2ME22
is an antiproliferative molecule that effectively induces apoptosis in actively proliferating cells in vitro and in vivo(1)
. It is an endogenous metabolite of E2 formed by the sequential hydroxylation and methylation at the 2-position
(2)
. 2ME2 is emerging as an attractive drug candidate because of its unique characteristics: (a) nontoxic
(3, 4)
; (b) p.o. available
(4,
5,
6,
7,
8,
9)
; (c) no effect on several responses normally associated with estrogens
(10,
11,
12,
13)
; and (d) selective inhibition of proliferating cells
(4, 14)
. The antitumor and antiangiogenic effects of 2ME2 are well reported, yet there is no known physiological function for this molecule. Several mechanisms of action have been proposed for 2ME2 activity, such as effects on tubulin polymerization and depolymerization
(15,
16,
17)
, up-regulation of p53
(14, 18)
, and death receptor 5
(19)
, and inhibition of superoxide dismutase enzymatic activity
(20)
. Although some of them are clearly sufficient to explain the activity of 2ME2 in certain cases, there is no evidence for a common mechanism of action operative in all of the cells sensitive to 2ME2.

A critical question for the development of this metabolite of E2 as a therapeutic agent is whether 2ME2 can engage ERs with a functional consequence. Previous studies have shown that at doses at which E2 and other estradiol metabolites are active, 2ME2 does not induce many of the activities associated with estrogens, such as uterine growth
(12, 13)
, induction of estrus phase in ovariectomized animals, reduction of seminal vesicles, and inhibition of leukopoiesis
(10)
. In addition, 2ME2 does not sustain tumor growth of a xenograft of H-301 cells, an estrogen-dependent hamster kidney cell line. In a model for estrogen-induced carcinogenicity, where several other estradiol derivatives are active, 2ME2 is not able to induce tumor formation
(11)
. The striking absence of functional estrogenic activity by 2ME2 under these conditions is consistent with its 2000-fold lower affinity than estradiol for the uterine cytosolic ER
(12)
. The affinity of 2ME2 for ERβ, a recently identified ER
(21)
has not been reported. In the present study we investigate the relationship between ERs and the mechanism of action of 2ME2 by determining the binding to recombinant human ERα and ERβ, by evaluating whether ER antagonists or agonists influence the activity of 2ME2, and by assessing whether at the appropriate concentration it may act as an agonist of ERs. Our findings indicate that 2ME2 is truly unique among E2 metabolites because there is no evident functional interaction between 2ME2 and the ERs, and the latter do not play a role in the antiproliferative mechanism of 2ME2.

MATERIALS AND METHODS

Chemicals.

The following chemicals were purchased: 2ME2 (Tetrionics, Madison, WI), E2 (Sigma, St. Louis, MO), ICI 182,780 (Tocris, Ballwin, MO), and galangin (Aldrich, St. Louis, MO). 2PE2 was the kind gift of Dr. Mark Cushman (Purdue University, West Lafayette, IN; Ref.
22
). Stock solutions (30 mm) of 2ME2, 2PE2, and galangin, and 10 mm stock solutions of E2 and ICI 182,780 were made in DMSO, aliquoted, and stored at −80°C. The compounds were diluted in incubation medium before each experiment, and the thawed stock solutions were discarded.

Receptor Binding Studies.

Binding studies were performed by displacement of [3H]estradiol from baculovirus-expressed recombinant human ERα and ERβ at 0.5 nm E2, using 1.0 μm diethylstilbestrol to calculate the nonspecific binding. The assays were performed by MDS Panlabs, Bothell, WA, under the conditions described in Obourn et al.(23)
. Ki values were calculated using the equation of Cheng and Prusoff
(24)
using the observed IC50 of the tested compound, the concentration of radioligand in the assay, and the historical value for the Kd of the ligand obtained experimentally at MDS Panlabs.

Proliferation Assays.

Proliferation was measured by cell counting using a Coulter Z1 cell counter (Coulter Corporation, Hialeah, FL) or by evaluation of DNA synthesis. Each condition was done in triplicate, and experiments were repeated at least twice. For MCF7 estrogen-dependent proliferation assay the cells were seeded in complete medium at 20–30,000 cells/well in a 24-well plate. After allowing the cells to adhere overnight the seeding density was determined by cell counts. Cells were washed with PBS (37°C), and starved by placing them in Improved Minimal Essential Medium-phenol red-free medium containing 2% charcoal-dextran fetal bovine-stripped serum (Georgetown University) and 1× antibiotic-antimycotic. After 3 days of starvation, cells were treated with or without increasing concentrations of compounds, replacing the medium every 2–3 days and counted after 8 or 10 days of treatment. To ensure that only the added test compounds stimulated growth, experiments were performed only with cell cultures that duplicated less than once during the starvation period.

For HUVEC and MDA-MB-435 proliferation assays, cells were plated at 20,000 cells/well. The next day cells were treated with or without increasing concentrations of compound for 72 h and then counted.

Detection of DNA synthesis was performed by use of the BrdUrd cell proliferation colorimetric ELISA kit from Roche (Indianapolis, IN) according to the manufacturer’s instructions. For BrdUrd assays, the cells were seeded at 5000 cells/well in a 96-well plate, allowed to attach overnight, and then exposed to the compound for 48 h.

Apoptosis Assays.

Apoptosis was measured by quantitation of cytoplasmic histone-associated DNA fragments using the cell death detection ELISA kit from Roche according to manufacturer’s instructions. Briefly, the cells were seeded in 24-well plates (20,000 MCF7 cells/well, 5,000 MDA-MB-435 cells/well, or 5,000 MDA-MB-231 cells/well), allowed to attach overnight, and then exposed to the appropriate drug for 96 h for MCF7 cells, and 48 h for MDA-MB-435 and MDA-MB-231 cells.

Inhibition of CYP450 Enzymes Activity.

Inhibition of baculovirus-expressed recombinant human CYP450 enzymes was performed by Gentest (Woburn, MA) using specific substrates. In brief, dealkylation of the specific substrates by the various enzymes was assessed in the presence and absence of galangin or a known specific inhibitor (positive control) after incubation with the appropriate cofactors (NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase). Enzymatic activity was followed by the increase in fluorescence of the dealkylated substrates. The following enzyme: substrate pairs were assessed: CYP1A1:7-benzoyloxyresorufin, CYP1B1:7-benzoyloxyresorufin, CYP1A2:3-cyano-7-ethoxycoumarin, CYP2C9:7-methoxy4-trifluoromethylcoumarin, CYP2C19: 3-cyano-7-ethoxycoumarin, and CYP2D6:3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin, and for CYP3A4, both reorufin benzyl ether and 7-benzyloxy-4-trifluoromethylcoumarin.

RESULTS

2ME2 Has Low Binding Affinity for ERα and ERβ.

Previous studies have shown that 2ME2 has low binding affinity for uterine cytosolic ER
(12)
. The recent identification of a second ER
(21)
prompted us to determine the binding affinity of 2ME2 for purified ERα and ERβ. Binding studies using recombinant proteins showed that 2ME2 has a Ki of 21 nm for ERα and a Ki of 417 nm for ERβ. Thus, 2ME2 has a 500- and 3200-fold lower affinity than estradiol for ERα and ERβ, respectively (Table 1)
⇓
. This confirms the low affinity of 2ME2 reported for ERα and demonstrates that the affinity for ERβ is even lower. Consistent with previous reports, E2, and the ER antagonists ICI 182,780
(25)
and tamoxifen
(26)
have binding affinities within the same order of magnitude.

Ki values were calculated as described in “Materials and Methods.” Results for E2, ICI 182,789, and Tamoxifen are from one experiment, while the results for 2ME2 are the average of three different experiments. In each experiment three independent binding assays were performed.

2ME2 Is Not an Agonist for ERs.

The results in Table 1
⇓
indicate that 2ME2 binds to ERs at nanomolar concentrations; at these concentrations the antiproliferative activity is not maximal. To examine whether 2ME2 could engage the ERs as an agonist and stimulate the proliferation of estrogen-dependent cells in serum stripped of steroids we used a MCF7 proliferation assay. The breast carcinoma cell line MCF7 (ERα+, ERβ+; Ref.
27
) requires estrogen stimulation for growth. Cells are allowed to achieve quiescence by culturing them in charcoal dextran-stripped serum and then incubating them with increasing concentrations of compound for several days; proliferation is determined by cell counts. The cells in the charcoal dextran-stripped serum do not proliferate and remain at approximately the seeding density throughout the assay period. Increasing concentrations of E2 result in a proliferative response with a peak stimulation at 1 nm E2 (Fig. 1A)
⇓
. When the cells are exposed to 2ME2 we detect a proliferative response starting at concentrations of 100 nm with maximal activity between 300 and 600 nm. At higher concentrations the antiproliferative activity of 2ME2 is evident. Under these assay conditions, maximal stimulation of proliferation by 2ME2 results in ∼40% of the maximal E2 proliferative response (Fig. 1C)
⇓
.

A number of E2 metabolites induce estrogenic responses, including 2HO-E2. Although the methylation of 2HO-E2 to 2ME2 by catechol-O-methyl transferase is essentially a nonreversible reaction, several tissues contain CYP450 enzymes capable also of demethylating 2ME2 back to 2HO-E2(2, 28,
29,
30)
. To determine whether the ability of 2ME2 to stimulate the proliferation of MCF7 cells is an inherent property of the 2ME2 molecule or is because of its metabolism by endogenous CYP450 enzymes, we evaluated the effect of CYP450 inhibitors on 2ME2-induced MCF7 proliferation. Flavonoids, a family of structurally related natural and synthetic polyphenol derivatives, include various compounds that have been shown to inhibit CYPs
(31)
. Several of them also exhibit proliferative and antiproliferative responses in estrogen-dependent and estrogen-independent cell lines
(32, 33)
. A number of inhibitors were tested, and among them galangin was selected because it did not inhibit the proliferative response induced by E2 (Fig. 1C)
⇓
. As shown in Fig. 1B⇓
, although 3 μm galangin by itself induces a slight proliferative effect on MCF7 cells, it significantly inhibits the ability of 2ME2 to induce cell growth. 2ME2 maximal proliferative response occurs at 600 nm, and this response is attenuated by 3 μm galangin (Fig. 1, B and C)
⇓
, and at 5 μm galangin the inhibition of 2ME2-stimulated growth is almost complete (Fig. 1C)
⇓
.

The concentrations of galangin that inhibit the proliferative response induced by 2ME2 are in the same range as the IC50 values of galangin for several well-characterized CYP450 enzymes (Table 2)
⇓
. Because galangin under these conditions did not inhibit E2-stimulated proliferation (Fig. 1C)
⇓
, its effects on 2ME2 are specific and not attributable to an impairment of the cells to proliferate. However, it was still possible that galangin-inhibited 2ME2 sustained proliferation by interfering with other processes specifically associated with 2ME2 stimulation, apart from inhibition of metabolism. To evaluate this possibility, we assessed the effect of galangin on the proliferation sustained by 2PE2, a closely related nonhydrolyzable analogue of 2ME2(22)
, which also stimulates proliferation of MCF7 cells at low concentrations and has antiproliferative effects at concentrations approaching the micromolar range (Fig. 1D)
⇓
. Galangin does not affect the response of this nonhydrolyzable analogue, indicating that contrary to 2ME2, the proliferative activity of 2PE2 is intrinsic to this molecule and suggesting that the metabolism in 2ME2 that renders it estrogenic may be associated with the 2-position. The IC50 value of 2PE2 as an antiproliferative agent is not affected by the CYP450 inhibitor (Fig. 1D)
⇓
. On the other hand, the shift to lower concentrations in the IC50 value of 2ME2 as an antiproliferative agent by galangin (Fig. 1B)
⇓
is consistent with the maintenance of a higher concentration of 2ME2 and/or a diminished production of a stimulatory metabolite, potentially 2HO-E2. In conclusion, the absence of a proliferative response, in the presence of a CYP450 inhibitor, at concentrations that are in the range of binding ERs suggests that 2ME2 cannot engage an ER as an agonist.

Inhibition was assessed with microsomes prepared from baculovirus-infected insect cells as described in “Materials and Methods.”

The Antiproliferative Activities of 2ME2 Do Not Require ER Expression and Are Not Influenced by ER Antagonists or Agonists.

We observe a comparable antiproliferative and apoptotic response after treatment with 2ME2 of the estrogen-responsive MCF7 cell line and of two cell lines that are estrogen-independent, MDA-MB-435 and MDA-MB-231 cells (Fig. 2, A and B)
⇓
. The IC50 values for the antiproliferative activities of 2ME2 are 1.5 μm, 1.3 μm, and 1.1 μm for MCF7 cells, MDA-MB-435 cells, and MDA-MB-231 cells, respectively. Furthermore, 2ME2 inhibits proliferation and induces apoptosis similarly in HeLa cells, which are ER-negative (data not shown; Ref.
34
). These results suggest that 2ME2 has antiproliferative activity irrespective of whether the cells are responsive to estrogens and of ER expression status.

2ME2 inhibits the proliferation of both tumor cells and endothelial cells. Despite being active in the absence of ERs, 2ME2 could still use ERs when present. To evaluate this possibility we assessed the effects of the ER antagonist ICI 182,780 on the antiproliferative activity of 2ME2 in the estrogen-independent cell line MDA-MB-435 and the estrogen-responsive cell type HUVEC. Both cell types express ERβ mRNA but not ERα protein (data not shown). Although we tried several commercially available antibodies, we could not detect expression of ERβ at the protein level in any of our cell types. ICI 182,780 has much greater affinity for ERs than 2ME2 (Table 1)
⇓
; at 10 μm ICI 182,780 has no effect on the proliferation of MDA-MB-435 (Fig. 3A)
⇓
and decreases the proliferation of HUVEC by 39% (Fig. 3B)
⇓
. However, the antiproliferative effects of 2ME2 are not affected by ICI 182,780 in either cell type (Fig. 3)
⇓
. This lack of an effect of ICI 182,780 on the antiproliferative activity of 2ME2 suggests 2ME2 acts independently of ERβ.

The ER antagonist ICI 182,780 does not interfere with the antiproliferative activity of 2ME2. MDA-MB-435 cells (A) and HUVEC (B) were treated with increasing concentrations of 2ME2 in the absence (•) or presence (○) of 10 μm ICI 182,780; no treatment (▪) or 10 μm ICI 182,780 without 2ME2 (□); bars, ±SD.

As a complementary approach, we assessed whether an ER agonist (E2) could affect 2ME2 antiproliferative activity. As shown above ICI 182,780 inhibits the proliferation of HUVEC (Fig. 3B⇓
and Fig. 4A⇓
). The addition of 0.1 μm E2 reduces this inhibition, and 0.7 μm E2 completely abrogates it (Fig. 4A)
⇓
. In contrast, these same concentrations of E2 do not influence the antiproliferative activity of 2ME2 on HUVEC (Fig. 4B)
⇓
. To evaluate the effect of E2 on 2ME2 response in a cell line expressing both ERs, we used MCF7 cells. These cells are estrogen-dependent and proliferate at very low concentrations of E2. Fig. 4C⇓
shows that increasing concentrations of E2 shift the IC50 values for ICI 182,780 to the right over several orders of magnitude. In contrast, similar increases of E2 have no effect on the IC50 values of 2ME2 (Fig. 4D)
⇓
. The experiments with 2ME2 were performed in the presence of 5 μm galangin to avoid an estrogenic response because of metabolism of 2ME2. Because galangin alone at this concentration induces a moderate proliferative response in MCF7 cells (Fig. 1B)
⇓
, in Fig. 4D⇓
proliferation is also observed in the absence of E2. As expected, the efficacy of an antiproliferative agent such as ICI 182,780, which acts as an antagonist of E2 for ER binding, is affected by increasing concentration of E2. The lack of such an effect of ICI 182,780 and E2 on 2ME2 activity under similar conditions indicates that even when ERs are expressed they do not play any role in the antiproliferative activity of 2ME2.

The antiproliferative activity of an ER antagonist, but not of 2ME2, is inhibited by estradiol in both HUVEC and MCF7 cells. HUVEC proliferation assays (A and B) were performed as described in “Materials and Methods.” •, no E2; ○, 0.1 μm E2; ▾, 0.7 μm E2. MCF7 proliferation assays (C and D) were performed as described in “Materials and Methods.” In D all samples contained 5 μm galangin. •, no E2; ○, 10 pM E2; ▾, 1 nm E2; ▿, 100 nm E2; bars, ±SD.

DISCUSSION

This study was aimed at assessing whether ERs are involved in the mechanism of action of 2ME2. 2ME2 is a natural metabolite of estradiol that in vitro inhibits actively proliferating tumor and nontumor cell lines in the high nanomolar to low micromolar concentrations. p.o.-administered 2ME2 inhibits angiogenesis, growth of primary tumor, and metastatic spread
(1)
. Therefore, 2ME2 is unique among the many antiangiogenic agents currently in clinical trials, because it targets not only the endothelial cell but also the tumor cell compartment of a growing tumor.

2ME2 is also unique among the major natural metabolites of estradiol, because it has the lowest affinity for the ERs and distinct biological activities
(12)
. In addition, the affinity of 2ME2 for ERβ is significantly lower than for ERα (Table 1)
⇓
. The majority of the molecules that bind the ERs bind both subtypes with similar affinities; however some of the synthetic E2 and naturally occurring steroidal ligands have different relative binding affinities for ERα compared with ERβ
(26, 35)
. 2ME2 is unusual in that E2 metabolism occurs mainly by oxidation at the A and D rings, and subsequent methylation by the ubiquitous catechol-O-methyltransferase enzyme
(2, 28)
. Several reports have investigated various estradiol metabolites for their ability to inhibit cellular proliferation, and whereas there are differences in their activities on the various cell types, 2ME2 consistently exhibits the greatest antiproliferative activity (Refs.
4
,
36
,
37
; data not shown). 2HO-E2 for comparison can act as an ER agonist but also has been shown to cause inhibition of cellular proliferation in smooth muscle cells, follicular granulosa cells, cardiac fibroblasts, endothelial cells, and tumor cell lines
(20, 38,
39,
40)
. We have also found that 2HO-E2 can inhibit the growth of tumor and endothelial cells in vitro, but in all of the cell types tested 2ME2 has greater antiproliferative effect. Conversely and in contrast to 2ME2 we did not observe apoptosis induced by 2HO-E2, suggesting a different mechanism for the inhibitory effects of these two metabolites of E2 (data not shown). Lottering et al.(41)
reported that exposure of cells to 2ME2 or 2HO-E2 resulted in different patterns of protein phosphorylation, additionally supporting the hypothesis that 2ME2 and 2HO-E2 use distinct signaling pathways. On the other hand, some reports suggest that the inhibitory activities associated with 2HO-E2 could be avoided by preventing its conversion to 2ME2(34, 42)
.

Interestingly, at high concentrations (>10 μm) E2 inhibits proliferation and induces apoptosis. These activities are not inhibited by ICI 182,780 (data not shown) suggesting that it is not through an ER-mediated pathway. However, it is unclear whether E2 at high concentrations is able to bind the molecular target for 2ME2 and if the antiproliferative activities of E2 are mediated through the 2ME2 pathway.

In the presence of the CYP450 enzymes inhibitor, galangin, 2ME2 cannot stimulate the proliferation of MCF7 cells, suggesting that it cannot engage ERs as an agonist. Furthermore, galangin inhibits numerous CYP450 enzymes in the range of concentrations at which it inhibits proliferation sustained by 2ME2 (compare Table 2
⇓
and Fig. 1
⇓
). Under similar conditions, the proliferation sustained by other agents is not affected (Fig. 1, C and D)
⇓
. These data are consistent with a metabolite of 2ME2 being responsible for the proliferative activity observed in the absence of galangin. NADPH-dependent CYP450 enzymes hydroxylate estradiol and can demethylate 2ME2, either process resulting in the production of 2HO-E2(29, 30)
. The different outcome that galangin has on the IC50 values for 2ME2 and 2PE2 is also consistent with a specific effect of galangin at the 2-position of 2ME2 (compare Figs. 1, B and D⇓
). However, attempts using high-performance liquid chromatography
(43)
or microsomes to identify 2HO-E2 in the supernatant or the cell extracts from cells cultured in the absence of galangin have not been successful. In preclinical toxicology studies in rats and dogs administered oral daily doses as high as 1800 mg/m2/day for 28 days, 2ME2 induced reversible hepatic changes in rats, and reversible estradiol-like changes to the reproductive system in both rats and dogs
(44)
. Interestingly, the in vivo estrogenic activity in different species correlates with the ability of liver microsomes of the same species to induce rapid and extensive conversion of 2ME2 into 2HO-E2 and 2-hydroxyestrone.
3
These data also suggest in vivo a correlation between the estrogenic activity of 2ME2 observed in some tissues at very high doses and its demethylation to 2HO-E2. The lack of intrinsic estrogenic activity of 2ME2 and its ability to inhibit cell proliferation independently of ERs indicate that it would be possible to generate analogs that could not be converted enzymatically into estrogenic derivatives and still maintain its antiproliferative activity.

E2 regulates a wide variety of physiological responses including activity on reproductive tissue, cardiovascular system, and bone metabolism. 2ME2 does share many of the activities of estrogens on the cardiovascular system and bone metabolism. 2ME2, like E2, may exert cardioprotective effects by increasing prostacyclin synthesis and by lowering cholesterol levels in serum
(45, 46)
, and at least some of the cardioprotective effects of 2ME2 have been shown to be ER-independent
(47)
. In intact young rats, 2ME2 treatment results in a reduction of the growth plate and the rate of longitudinal bone growth. E2 treatment of intact young rats results in a similar activity; however, E2 but not 2ME2 treatment results in inhibition of radial bone growth and cancellous bone turnover
(46)
. On the other hand, in ovariectomized rats 2ME2 mimics all of the effects of E2 on bone metabolism but at concentrations 10–100-fold lower than what is necessary for optimal inhibition of tumor growth.
4
Interestingly, the cardioprotective and bone metabolism effects of 2ME2 are observed at doses that show low antitumor activity and do not produce the reversible E2-like activities on reproductive tissue observed in the preclinical toxicology studies
(1, 45)
.
4
Several studies suggest that the E2 responses on the reproductive and cardiovascular systems are mediated through the classical nuclear receptors
(45, 48, 49)
. The effects of 2ME2 on bones and cholesterol levels resemble the activity of selective estrogen response modifiers
(50)
. However, contrary to the selective estrogen response modifiers, 2ME2 has much lower affinity and does not engage the ERs. Additionally, 2ME2 does not bind other steroid nuclear receptors with high affinity (data not shown). Our studies have not addressed whether 2ME2 engages any of the nuclear orphan receptors. This is particularly interesting because it has been shown recently that the synthetic estrogen diethylstilbestrol interacts with the orphan nuclear receptor ERRs, and this has a functional consequence on the differentiation of trophoblast cells
(51)
. The ERRs were identified based on their sequence homology to the ERα. ERRs are not activated by natural estrogens and their activities are not influenced by antiestrogens. Additional studies are required to determine whether 2ME2 is engaging these receptors.

Collectively, in this study we show that 2ME2 is not an agonist for the ERs and demonstrate that its antiproliferative activity is mediated independently of ERα and ERβ.

Acknowledgments

We thank Drs. Tekum Fonong and James Yager at Johns Hopkins University, Baltimore, MD, for performing high-performance liquid chromatography analysis of cell culture medium and Sauda Ayub for help in preparing the figures. We also thank Drs. Anthony Treston and Royce Mohan for critical reading of the manuscript.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.